The final step of dietary iron assimilation - the exsorption of iron from the mucosa into circulation - involves the iron transport protein ferroportin. Macrophages of the reticuloendothelial system also play a major role in iron metabolism by recycling iron from damaged or senescent erythrocytes. Ferroportin also plays a major role in the release of iron from macrophages, and thus provides the major mechanism for the export of iron from the cells of our body. Body iron stores, hypoxia, the rate of erythropoiesis, pregnancy and inflammation all have dramatic effects on dietary iron assimilation and iron recycling by the reticuloendothelial system. How such factors influence the activity of ferroportin to modify iron export is an active area of interest in the field. The most important recent discovery has been the role of the liver-derived peptide hepcidin. Hepcidin is produced under iron- loading and inflammatory conditions to suppress dietary iron absorption and macrophage iron recycling, and its synthesis is diminished in response to iron deficiency, pregnancy or enhanced erythropoiesis to promote iron uptake from the diet and enhance iron recycling. This peptide therefore provides a direct link between the liver, bone marrow and intestine to adjust metabolism to meet the body's demand for iron. Hepcidin regulates ferroportin protein levels through binding interactions that induce its internalization and lysosomal degradation. The model that ferroportin is a receptor for hepcidin implicates a homeostatic mechanism that allows systemic regulation of intestinal iron absorption and macrophage iron recycling directly in response to the body's iron demands targeting ferroportin function. This model is borne out by clinical studies of hemochromatosis, an inherited disorder leading to abnormal iron accumulation in different tissues. This disease is associated with mutations in genes encoding HFE, hepcidin, hemojuvelin, transferrin receptor-2 or FPN. Although the molecular pathogenesis has not been precisely elucidated in all cases, abnormal regulation of iron metabolism by hepcidin has been proposed as a common, unifying feature of the different forms of this disease. The specific aim of this R21 proposal is to develop a high throughput assay to screen for small molecule inhibitors of hepcidin-ferroportin binding interactions. Our strategy is to utilize Sfp-catalyzed site-specific protein labeling to establish a rapid, sensitive, and reproducible fluorescent-based screen for small molecules that block ligand-induced degradation of the hepcidin receptor, ferroportin. The project has broader applicability in the development of HTS to assay ligand-receptor interactions as well as representing a major advance towards the discovery of small molecule probes of iron transport.